The long-term objective of this project is a therapeutic adjunct to prevent chemotherapy-induced diarrhea (CID) based on a completely new mechanism of action targeting enteric bacteria. This Fast-Track SBIR proposal outlines the strategy for selecting a lead development candidate to be evaluated in investigational new drug (IND)-enabling studies to support a first-in-human trial. The focus of this project is prevention of diarrhea associated with irinotecan (IRI), an important drug used to treat advanced colorectal and pancreatic cancer patients. Intense, delayed diarrhea is the major reason for reducing, postponing or stopping IRI chemotherapy. The cancer-killing, active agent of IRI is SN38, a potent topoisomerase I inhibitor. As part of its elimination from the body, SN38 is detoxified primarily by the liver to an inactive glucuronide (SN38-G) that is subsequently shuttled to the lower gastrointestinal tract. The ?-glucuronidase enzyme (GUS) expressed in a subset of gut bacteria metabolizes SN38-G back into SN38, which is highly toxic to enterocytes. This reactivation of SN38 by bacterial GUS in the gut microbiome is a key triggering event leading ultimately to serious, delayed diarrhea. A selective, non-proprietary bacterial GUS inhibitor (SBX-1) was previously shown to alleviate IRI-induced diarrhea in rodents. Phase 1 of this project discloses five proprietary analogs of SBX-1 and outlines the efficacy/tolerability studies to enable selection of the two most promising analogs to advance into Phase 2 SBIR studies. The lead candidate will be selected in the first aim of Phase 2. The remaining Phase 2 activities include chemistry/manufacturing/control (CMC)-related work and regulatory/safety preclinical studies that will transition this project from drug discovery to preclinical development. Aim 1 (Phase 1): Profile novel SBX-1 analogs in vitro for their off-target pharmacology, cytotoxicity, metabolism liability and stability in plasma. Aim 2 (Phase 1): Identify the two most promising SBX-1 analogs based on their therapeutic window and candidacy for formal preclinical studies. Aim 3 (Phase 2): Select lead candidate. Generate CMC and additional non-GLP preclinical data to support a pre-IND meeting with the FDA. Aim 4 (Phase 2): Conduct CMC-related activities to enable formal evaluations of the formulated lead candidate in GLP toxicology and safety pharmacology studies. A key deliverable in the proposed Phase 2 work is a preclinical data package that will adequately qualify the lead candidate for further evaluation in an acute-administration, first-in-human clinical trial in advanced colorectal and/or pancreatic cancer patients undergoing irinotecan-containing chemotherapy.